Regulation of methanol utilisation pathway genes in yeasts

Abstract

Methylotrophic yeasts such as Candida boidinii, Hansenula polymorpha, Pichia methanolica and Pichia pastoris are an emerging group of eukaryotic hosts for recombinant protein production with an ever increasing number of applications during the last 30 years. Their applications are linked to the use of strong methanol-inducible promoters derived from genes of the methanol utilisation pathway. These promoters are tightly regulated, highly repressed in presence of non-limiting concentrations of glucose in the medium and strongly induced if methanol is used as carbon source. Several factors involved in this tight control and their regulatory effects have been described so far. This review summarises available data about the regulation of promoters from methanol utilisation pathway genes. Furthermore, the role of cis and trans acting factors (e.g. transcription factors, glucose processing enzymes) in the expression of methanol utilisation pathway genes is reviewed both in the context of the native cell environment as well as in heterologous hosts.

Figure 1

Methanol utilisation pathway in methylotrophic yeasts: The main pathways and the respective enzymes working in the methanol metabolism in methylotrophic yeasts are shown. AOX: alcohol oxidase (EC 1.1.3.13), CAT: catalase (EC 1.11.1.6), FLD: formaldehyde dehydrogenase (EC 1.2.1.1), FGH: S-formylglutathione hydrolase (EC 3.1.2.12), FDH: formate dehydrogenase (EC 1.2.1.2), DAS: dihydroxyacetone synthase (EC 2.2.1.3), TPI: triosephosphate isomerase (EC 5.3.1.1), DAK: dihydroxyacetone kinase (EC 2.7.1.29), FBA: fructose 1,6-bisphosphate aldolase (EC 4.1.21.13), FBP: fructose 1,6-bisphosphatase (EC 3.1.3.11), MFS: methylformate synthase; DHA: dihydroxyacetone, GAP: glyceraldehyde 3-phosphate, DHAP: dihydroxyacetone phosphate, F_1,6BP: fructose 1,6-bisphosphate, F₆P: fructose 6-phosphate, P_i: phosphate, Xu₅P: xylulose 5-phosphate, GSH: glutathione, PYR: pyruvate; PPP: pentose phosphate pathway, TCA: tricarboxylic acid cycle

Figure 2

Regions within methanol utilisation pathway gene promoters identified as cis-acting elements: The regions indicated in this figure were identified either by deletion from the original promoter and proving there activity in a different promoter environment, by footprinting and/or by bandshift assays. HpMOXp: H. polymorpha MOX promoter [88, 122, 123], PpAOX1p: P. pastoris AOX1 promoter [125, 126], PpAOX2p: P. pastoris AOX2 promoter [93], CbFDH1p: C. boidinii FDH1 promoter [128]; URS1, URS2: upstream repressing sequences, UAS, UAS1, UAS2: upstream activation sequences, UAS-M, UAS-FM: upstream activation sequence for methanol and for methanol and formate, respectively, Region A: region upstream of -690, Region C: region between -540 and -400.